The invention provides improvements in the field of animal models for testing effects of genes introduced into animal cells or tissue by adenoviral gene transfer.
Adenoviruses infect cells using two cell surface receptors, the xe2x80x9cCoxsackie B and adenovirus 2 and 5 receptorxe2x80x9d (hereinafter referred to as CAR; Bergelson J. M., et al, Science 275, 1320-23, 1997) and the integrin receptors (xcex1xcexdxcex23 or xcex1xcexdxcex25; Wickham, T. J. et al, Cell 73, 309-19, 1993) the contents thereof being incorporated herein by reference. Adenoviral based vectors are widely used in gene therapy, as they represent one of the most efficient ways to deliver genes to target cells. They are of particular interest for in vivo gene therapy proof-of concept experiments in rodent models. However, rodent tissues are not well transducible with adenoviral vectors.
In its broad aspect the invention is concerned with genetic modification of target cells which are normally refractory to adenoviral transduction. More particularly the invention provides a plasmid construct that expresses a porcine adenovirus receptor (PCAR) and transgenic animals that show expression of pCAR.
Organ transplants of liver, kidney, lung and heart are now regularly performed as treatment for endstage organ disease. Despite the use of modern immunosuppressive drugs acute and chronic graft (tissue or organ) rejection still remain major factors in graft loss. There is, therefore, a continued need for means to inhibit acute and chronic graft rejection and increase graft acceptance, e.g. through induction of peripheral tolerance without causing serious toxic side effects typically associated with conventional immunosuppressant therapy. When considering cell transplantation, e.g. bone marrow derived cells, islet cells, neuronal cells etc. one is faced with similar problems of rejection. Making organs or cells less immunogenic through genetic modification is seen as an alternative or add on to conventional immunosuppression.
Rodent animal models are of crucial importance for testing the immunomodulatory effects of new gene products. However in the case of using adenovirus as gene delivery vehicle rodent models have so far proven to be of limited value, as many rodent organs or cell types are refractory to adenoviral transduction. This may be due to the fact that either the adenoviral receptor CAR is not expressed or only weakly expressed on the cell surface of the cells of interest.
Accordingly, the invention provides a plasmid or vector construct that comprises a DNA molecule which expresses porcine CAR (SEQ ID NO:4 hereinafter referred to as pCAR) or a biologically active fragment or derivative thereof, for example a C-terminally truncated porcine CAR (SEQ ID NO:2hereinafter referred to as xcex94pCAR), that retains full functionality as adenoviral receptor.
pCAR comprises an intracellular domain, a transmembrane domain and a an extracellular domain that binds to the adenoviral fibre proteins, i.e. a total sequence of 365 amino-acids. It will be understood that any nucleic acid sequence encoding a porcine CAR homologue is a candidate for utilization in the present invention. For example, it may include a pCAR sequence with a modified, mutated or truncated region thereof, that retains the activity of mediating adenoviral transduction. It will be further understood by the skilled person that any nucleic acid sequence which encodes a biologically active form of pCAR, including but not limited to a genomic or cDNA sequence or functionally equivalent variant or mutant thereof or a fragment thereof which encodes a biologically active protein fragment or derivative which mediates adenoviral transduction, may be utilized in the present invention. For example, xcex94pCAR may comprise the leader sequence of 19 amino-acids, the extracellular domain of 216 amino-acids, the transmembrane domain of 24 amino-acids and a truncated cytoplasmic domain, e.g. limited to 3 amino-acids. Two potential sites for N-glycosylation are located at Asn 106 and Asn 201. Amino-acids present in the sequence which are not essential to the activity may be changed by mutation, e.g. amino-acid 258 may be changed from Val to Ile; amino-acid 262 may be changed from His to Arg.
Preferred nucleic acid sequence for use in the invention is e.g. as disclosed in SEQ ID NO: 1 from nucleotide 3229 to nucleotide 4014. The corresponding amino acid sequence encoded by such DNA sequence is indicated in SEQ ID NO:2.
Any known expression vector or plasmid that is capable of expression upon transfection of a specified eukaryotic target cell may be utilized to pratice the invention. xe2x80x9cPlasmidxe2x80x9d and xe2x80x9cvectorxe2x80x9d can be used interchangeably in the present specification as the plasmid is the most commonly used form of vector. An expression vector is a vector capable of directing the expression of genes to which they are operatively linked. An operable linkage as used herein refers to the position, orientation and linkage between a structural gene and expression control element(s) such that the structural gene can be expressed in any host cell. The term xe2x80x9cexpression control elementxe2x80x9d includes promoters, enhancers, ribosome binding sites etc. Any eukaryotic promoter and/or enhancer sequences available to the skilled person which are known to control expression of the nucleic acid of interest may be used in plasmid vector constructs, including but not limited to a cytomegalovirus (CMV) promoter, a Rous Sarcoma (RVS) promoter, a Murine Leukemia (MLV) promoter, a herpes simplex virus (HVS) promoter, such as HSV-tk, a xcex2-actin promoter, e.g. chicken xcex2-actin, as well as any additional tissue specific or signal specific regulatory sequence that induces expression in the target cell or tissue of interest. A preferred expression vector or plasmid according to the invention is e.g. an eukaryotic expression vectors, e.g. a pxcex2-actin-p16PL vector such as p(chicken)xcex2-actin-p16PL.
In one such embodiment, a DNA sequence encoding pCAR is subcloned into the DNA plasmid expression vector, e.g. pxcex2-actin-p16PL, resulting in pxcex2-actin-pCAR-p16PL. p16PL is a standard mammalian expression vector, containing a gene that encodes a selectable marker, e.g. an antibiotic resistance gene, and a xcex2-actin promoter active in mammalian cells (K. M. Marsden et al, J. Neurosc., May 15, 1996, 16(10): 3265-3273). Such a construct, which may be constructed by one of ordinary skill with components available from numerous sources, will drive expression of a pCAR DNA fragment ligated downstream of the xcex2-actin promoter subsequent to transfection of the target cell. More specifically, pCAR is cloned from pig liver RNA using a PCR based approach. The PCR fragment is inserted into the expression vector pSport (Life Technologies). This plasmid serves as template to create the truncated version of xcex94pCAR. Preferably pxcex2-actin is pxcex2-(chicken) actin.
The invention further provides host cells into which a recombinant expression vector of the invention has been introduced. A host cell can be any prokaryotic or eukaryotic cell, e.g. bacterial such as E. Coli, yeast or mammalian cells, e.g. CHO or COS cells.
The host cells of the invention may preferably be used to produce nonhuman transgenic animals, preferably a mammal, more preferably a rodent such as a rat or mouse, or a pig.
For example, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a pCAR-coding sequence has been introduced. A transgenic animal of the invention, more preferably a mammal, most preferably a rodent or a pig, may be created by introducing a pCAR expression construct into the male pronuclei of a fertilized oocyte, e.g. by microinjection, or into embryonic stem cells, e.g. by electroporation. Methods for generating transgenic rodents have become conventional in the art and are described e.g. in U.S. Pat. Nos. 4,736,866, 4,870,009, 4,873,191, or in Manipulating the Mouse Embryo, B. Hogan, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). For example the expression construct may be introduced into an embryonic stem cell line and cells in which the introduced pCAR gene has integrated are selected. The selected cells are then used to produce chimaeras with known standard procedures. A chimaeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. The pCAR expression plasmid may also be inserted into somatic/body cells of the donor animal to provide a somatic recombinant animal, from whom the DNA construct is not capable of being passed on to offspring. For example, a somatic cell from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g. through the use of electrical pulses, to an enucluated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring of this female foster animal will be a clone of the animal from which the somatic cell is isolated. Or for example, an improved method of cloning pigs using donor nuclei from non-quiescent differentiated cells in which the desired DNA, e.g. porcine CAR or fragments or variants thereof, is inserted in said differentiated pig cell or pig cell nucleus. This improved method is described in U.S. Pat. No. 6,235,969 B1 and is hereby incorporated by reference.
The present invention also provides a method for improving adenoviral gene transfer in a rodent using a transgenic rodent which expresses or overexpresses pCAR. Such rodents may be used as models in gene therapy to test adenoviral transduction, e.g. prevention or treatment of acute or chronic graft rejection, autoimmune disorders, e.g rheumatoid arthritis, cardiovascular disorders, e.g. restenosis, nervous system disorders, e.g. parkinson disease, etc. A preferred embodiment of the invention is the use of such rodents expressing or overexpressing pCAR in transplantation experiments, for example, of organs, tissues or cells, e.g. lung, heart, kidney, liver, pancreas, small bowel, spleen, pancreatic islets, neuronal or stem cells, etc. For example, organs, tissues or cells of such transgenic rodents, e.g. mice, are removed, in vitro transduced with the adenoviral gene delivery vector to be tested and then transplanted into rodents, e.g. mice, e.g. such animals which do not express pCAR.
The functional expression of pCAR, e.g. xcex94pCAR may also be used to generate transgenic pigs that overexpress this adenoviral receptor. Porcine organs, tissues or cells transgenically modified to express high levels of pCAR may be used as recipients for adenoviral gene therapy vectors. Such transgenic modified organs, tissues or cells can be transfected with adenoviral gene therapy vectors carrying thrapeutically beneficial genes either ex vivo or in vivo and can be subsequently transplanted in a recipient. Beneficial genes are those that are expected to confer graft protection following transplantation of these gene delivered organs in xenotransplantation therapy. The present invention comprises a method to generate such transgenic pigs expressing high levels of pCAR or a functionally equivalent variant or mutant thereof or a fragment thereof, e.g. as disclosed above, and gene therapy methods for preventing or inhibiting graft rejection in a recipient using organs, tissues or cells of such transgenic pigs.
Definitions
To facilitate understanding of the invention, a number of terms are defined below.
xe2x80x9cFragmentxe2x80x9d of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. xe2x80x9cFragmentxe2x80x9d of a polynucleotide sequence refers to a polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO:2 and 4.
xe2x80x9cTransductionxe2x80x9d Transfer of genetic material or characteristics from one bacterial cell to another by the incorporation of bacterial DNA into a bacteriophage.
xe2x80x9cVariantxe2x80x9d refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gln-I Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like. Embodiments include methylation of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C-terminal glycines.
The following Examples are illustrative only and not limiting of the invention. The xcex2-actin promotor used in the Examples is the xcex2-(chicken)actin promotor.